Closing Domestic Nutrient Cycles Using Microalgae - ACS Publications

Subscriber access provided by University of Florida | Smathers Libraries

Article

Closing domestic nutrient cycles using microalgae Tania Vasconcelos Fernandes, Rabin Shrestha, Yixing Sui, Gustavo Papini, Grietje Zeeman, Louise E.M. Vet, Rene H. Wijffels, and Packo Lamers Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b02858 • Publication Date (Web): 21 Sep 2015 Downloaded from http://pubs.acs.org on September 21, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 19

Environmental Science & Technology

1

Closing domestic nutrient cycles using microalgae

2 3

Tânia Vasconcelos Fernandes*, Rabin Shrestha1, Yixing Sui1, Gustavo Papini1, Grietje Zeeman3,

4

Louise E.M. Vet4, Rene H. Wijffels2,5, Packo Lamers2

5 6

*

7

Wageningen, The Netherlands. (Phone: +31-317-473533; e-mail: [email protected])

8

1

9

Wageningen, The Netherlands.

Department of Aquatic Ecology, Netherlands Institute of Ecology (NIOO-KNAW), P.O. Box 50, 6700 AB

Department of Aquatic Ecology, Netherlands Institute of Ecology (NIOO-KNAW), P.O. Box 50, 6700 AB

10

2

11

Netherlands.

12

3

13

Netherlands.

14

4

15

Wageningen, The Netherlands.

16

5.

Bioprocess Engineering, AlgaePARC, Wageningen University, P.O. Box 16, 6700 AA Wageningen, The

Sub-department of Environmental Technology, Wageningen University, P.O. Box 8129, 6700 EV Wageningen, The

Department of Terrestrial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), P.O. Box 50, 6700 AB

University of Nordland, Faculty of Biosciences and Aquaculture, N-8049, Bodø, Norway

17 18

TOC ABSTRACT ART

19 1 ACS Paragon Plus Environment


Page 2 of 19

20 21

ABSTRACT

22

This study demonstrates that microalgae can effectively recover all P and N from anaerobically treated

23

black water (toilet wastewater). Thus enabling the removal of nutrients from the black water and the

24

generation of a valuable algae product in one step. Screening experiments with green microalgae and

25

cyanobacteria showed that all tested green microalgae species successfully grew on anaerobically

26

treated black water. In a subsequent controlled experiment in flat-panel photobioreactors, Chlorella

27

sorokiniana was able to remove 100% of the phosphorus and nitrogen from the medium. Phosphorus

28

was depleted within 4 days while nitrogen took 12 days to reach depletion. The phosphorus and

29

nitrogen removal rates during the initial linear growth phase were 17 mg.l-1.d-1 and 122 mg.l-1.d-1

30

respectively. After this initial phase, the phosphorus was depleted. The nitrogen removal rate

31

continued to decrease in the second phase, resulting in an overall removal rate of 80 mg.l-1.d-1. The

32

biomass concentration at the end of the experiment was 11.5 g.l-1, and had a P content of

33

approximately 1% and an N content of 7.6%. This high algal biomass concentration, together with a

34

relatively short P recovery time, is a promising finding for future post-treatment of black water while

35

gaining valuable algal biomass for further application.

36 37

Keywords

38

Nutrients recovery, Chlorella sorokiniana, toilet wastewater, phosphorus, nitrogen

39 40

INTRODUCTION

41

As the world’s population increases so does the amount of wastewater produced. Wastewater has

42

always been characterized as a polluted stream and therefore the 20th and 21st century wastewater

43

treatment plants focus on the removal of the organic and inorganic pollutants. However, modern

44

society has realized that these organic and inorganic compounds are actually valuable products that 2 ACS Paragon Plus Environment

Page 3 of 19


45

can be recovered and recycled back into our resource demanding society. In current centralized

46

sanitation systems urine and faeces are greatly diluted with the remaining household wastewater,

47

rainwater and small-scale industrial wastewater, therefore making it harder to recover its valuable

48

compounds. Hence, the current challenge is to rethink our present wastewater treatment system and

49

provide the technology to not only remove organic and inorganic compounds, but also recover them in

50

a sustainable and highly efficient manner.

51

New sanitation concepts where black water (BW - toilet wastewater) and grey water (GW -

52

shower/washing wastewater) are separated at the source, conserve organic and inorganic compounds

53

in a smaller volume which facilitates recovery1. The Netherlands Institute of Ecology (NIOO-KNAW)

54

in the Netherlands, has implemented such source separated system in its new building. The BW is

55

vacuum collected using only 1L of water per flush and then treated in a UASB (upflow anaerobic

56

sludge blanket), where the carbon is converted into biogas – a green energy source. Anaerobic

57

treatment of concentrated black water is proven technology for treating high-strength wastewater 2.

58

The remaining effluent, known as anaerobically treated black water (AnBW), contains the major part

59

of the nutrients. One of these nutrients is phosphorus. The world’s main source of phosphorus is

60

phosphate rock which, at the current extraction rate, will be depleted in the coming century3. Of all

61

phosphorus and nitrogen produced in a household’s wastewater, urine contains 40% of the phosphorus

62

and 69% of the nitrogen, while faeces contains 28% of the phosphorus and 13% of the nitrogen4. If

63

one is able to recover all the phosphorus from faces and urine, human excreta could supply 22% of the

64

world phosphorus demand5.

65

Phosphorus can be recovered from concentrated wastewater by struvite precipitation. However this

66

process requires a high pH ( > 8) and extra addition of magnesium to form the mineral6. Moreover,

67

even though struvite incorporates nitrogen, the amount of nitrogen that is recovered via this

68

technology is limited. A very promising alternative to recover phosphorus, and nitrogen at the same

69

time, is by microalgae growth.

3 ACS Paragon Plus Environment


Page 4 of 19

70

Microalgae are becoming increasingly interesting as renewable energy sources due to their fast growth

71

and non-competitive nature with regard to food production7. When sufficient light and carbon dioxide

72

are supplied, microalgae biomass production and nutrient uptake are high. The algae biomass could be

73

directly used as a fertilizer8, therefore returning the nutrients back to the soil to grow crops and thereby

74

closing the nutrient cycle. The algae biomass could also be used as a resource for a diversity of added-

75

value products.

76

Most studies on N and P removal by microalgae deal with low nutrient concentrations, either from

77

drinking water, fresh water, sewer or from waste streams, such as animal manure, that were diluted

78

prior to cultivation9-13. The choice of diluting is justified by the difficulty of growing microalgae at

79

high ammonia concentrations, which has been reported to inhibit microalgae growth14. However, when

80

the pH is kept neutral, ammonia inhibition is prevented, and growth of algae on the nutrient-rich

81

stream is possible, resulting in high biomass concentrations. The high biomass concentration is an

82

economic advantage as it reduces the overall costs of harvesting, which is one of the main reasons why

83

microalgae growth on wastewater is still not applied at full-scale15-17. Recently, one study has shown

84

growth of microalgae on nutrient-rich human urine. However, for higher nitrogen removal rates,

85

adjustment of the N:P ratio and addition of Magnesium was necessary 18. AnBW has higher

86

concentrations of magnesium than urine and a more favourable N:P ratio for algae growth (~ 45:1 for

87

urine vs ~30:1for AnBW). The high N:P ratio of AnBW is still higher than the 16:1 N:P ratio usually

88

reported for phytoplankton19 and so the challenge remains to reach full N and P removal. In addition,

89

the darker colour of the AnBW, which is attributed to the humic matter, has been reported to affect

90

microalgae growth both negatively, by absorbing part of the available light, and positively, by serving

91

as energy source and stimulating better growth20, 21.

92

The goal of the present work is to explore the potential of microalgae for phosphorus and nitrogen

93

recovery from AnBW and present a proof of principle that microalgae cultivation can be added as a

94

post-treatment technology after anaerobic treatment.

95


Page 5 of 19


96

MATERIALS AND METHODS

97

Culture medium

98

The culture medium used for microalgae growth was AnBW from a UASB reactor fed with vacuum-

99

collected black water, operated in Sneek, The Netherlands at 25°C and 8.3 days HRT. All containers

100

of AnBW were stored at 4°C until use. The average characteristics of the AnBW are presented in

101

Table 1.

102

Table 1. Average characteristics of the AnBW used for the experiments. Total nitrogen

-1

Total phosphorus (mgTP·L )

pH

7.6±0.2

Phosphate

Ammonia

(gTN·L-1)

(gNH4+-N·L-1)

1.21±0.01

1.07±0.08

(mgPO4-P ·L-1) 105.2±3.9

73±10

103

Data expressed as mean of three different time points of AnBW ± standard deviation.

104

The translucent brownish colour of AnBW does not negatively affect the microalgae cultivation, as is

105

often suggested22, because the light attenuation coefficient is much higher for a typical outdoor

106

microalgae culture (between 200-2000 m-1 in clear growth medium, depending on the microalgae

107

concentration) than for AnBW (between 70-140 m-1, depending on the amount of suspended solids of

108

AnBW). This means that at high density microalgae cultures, most of the light will be absorbed by the

109

microalgae cells and not by the suspended solids.

110

On the other hand, AnBW’s naturally high calcium carbonate (~ 4.6 g CaCO3.l-1) and high ammonia

111

concentration (~1gNH4+-N·L-1) do pose challenges for microalgae cultivation and demand for tight pH

112

control. More specifically, the high carbonate concentration requires constant aeration with high CO2

113

concentrations. Otherwise, the carbonate in the AnBW would leave the reactor via the gas stream in

114

the form of CO2 (to restore chemical equilibrium between dissolved carbon species and gaseous CO2),

115

thereby causing an increase in pH. Next to a direct negative impact of high pH on microalgal growth,

116

such a pH increase would cause the dissociation of the ammonium ion (NH4+) to toxic NH3. Due to the

117

high ammonia concentration of the AnBW, an NH3 concentration higher than the threshold value of 30 5 ACS Paragon Plus Environment


Page 6 of 19

118

mg NH3-N·l-1 reported for inhibition of green microalgae growth14 will be reached at pH higher than

119

7.7 for 25°C or 7.3 for 37°C. Therefore, in all experiments the pH was tightly controlled to neutrality

120

and the reactors were aerated with pressurized air enriched with 3-10% CO2 depending on the

121

experimental setup.

122

Microalgae species

123

Chlorella vulgaris UTEX 26, Scenedesmus obliquus CCAP276/3a, Synechococcus elongates SAG 89-

124

79, Anabaena flos-aquae CCAP1446/1C and Synechocystis sp. 6803WT were obtained from the algal

125

collection of NIOO-KNAW. Chlorella sorokiniana CCAP 211/8K was obtained from CCAP (Oban,

126

UK). All cultures were maintained under aseptic conditions in a WC medium 23 and incubated (Sanyo,

127

MLR-350) at 18°C with 14 h illumination (100 µmol·m-2·s-1) per day.

128

Experimental set-up for microalgae screening

129

The six different microalgae species were cultivated in batch-mode in 300 ml Erlenmeyer flasks

130

placed in an incubator with a shaking platform (Sanyo Gallenkamp PLC). The incubator was operated

131

at 25°C, 100 rpm. The incubator was aerated with 3% CO2 enriched air at a rate of 0.5 L·min-1. All

132

experiments were carried out in triplicate.

133

The pH of the AnBW was kept at neutrality by the addition of 75 mM HEPES buffer that was added

134

before algal inoculation. Moreover, pH was measured daily with a pH meter (WTW- 330i) and

135

deviation from neutrality was kept lower than 0.2 units with the addition of either 1M HCl or 1M

136

NaOH. Each flask was filled with 120 ml of AnBW and inoculated with 10000 cells·ml-1 of one of the

137

six algae species. After the inoculation, the flasks were placed and kept in the incubator throughout the

138

whole experiment. The flask positions in the incubator were daily randomly rotated in order to provide

139

an equal average light intensity of 175 µmol·m-2·s-1 to each flask. Samples were taken every 2-3 days

140

to follow the algal growth and the nutrient depletion.

141

Experimental set-up for microalgae N and P removal in controlled photobioreactors


Page 7 of 19


142

Three flat panel photobioreactors (PBRs)24 were operated under batch conditions as replicates. Each

143

PBR contained a total liquid volume of 380 ml, a light path of 14 mm and a total illuminated area of

144

0.027 m2. The content was homogeneously mixed by air bubbling enriched with 10% CO2 at a flow of

145

400 ml.min-1 (40 ml.min-1 CO2 and 360 ml.min-1 air). Each PBR was filled with AnBW and inoculated

146

with 175,000 cells·ml-1 of Chlorella sorokiniana. Temperature was kept at 37°C and pH at 7.0±0.7,

147

which are the optimal growth conditions for Chlorella sorokiniana25. The light intensity, averaged

148

over the entire culture volume (see calculations section), was kept at 100 µmol·m-2·s-1 for the first 5

149

days and at 150 µmol·m-2·s-1 for the remaining experimental time.

150

Chemical analyses

151

Samples for NH4+-N, PO43--P, NO3--N and NO2--N analysis were centrifuged (Sartorius 1-15P) at

152

14000 rpm for 10 minutes. The supernatant was filtered through a 0.2 µm cellulose acetate filter

153

(chroafil® ca-20/25) and measured in a Seal QUAATRO Auto Analyzer (Beun de Ronde, Abcoude)

154

according to standard methods. 26 Chlorophyll a fluorescence was measured with a Phyto-PAM (Heinz

155

Walz, Effeltrich, Germany) and algal biovolume (algal hold-up) with a CASY®1 Model TTC system

156

(Schärfe System GmbH). At the end of the experiment, algal dry weight (DW) and elemental

157

composition of the algal biomass (CNP) was determined according to standard methods.26 Elemental

158

composition was measured with an Elemental Analyzer (Flash 2000, Interscience Breda) and a Seal

159

QUAATRO Auto Analyzer.

160

Calculations

161

Free ammonia (NH3) was calculated according to Fernandes et al. (2012)27. Average light intensity

162

(Iavg) inside the entire culture was calculated according to Santos et al. (2014)28. The biomass yield on

163

light (Yx,E) in g.mol photon-1 during any given time interval was calculated by dividing the total

164

amount of biomass produced during that time interval by the total amount of light falling on the

165

photobioreactor in the same time interval, as described by Cuaresma et al.(2011)29. Only photons from

166

the photosynthetically active radiation (PAR) are taken into account in this publication. The amount of

167

nitrogen and phosphorus taken up by the microalgae was calculated based on the measured amount of



Page 8 of 19

168

dry algal biomass produced at the end of the experiment and on the N and P mass percentages of this

169

biomass. The amounts of nitrogen and phosphorus that were removed from the AnBW were calculated

170

as the concentration difference between the start and end of the experiment, for NH4-N and PO4-P in

171

the culture supernatant respectively. In cases where the removed amounts of N and P were compared

172

with the amounts of N and P taken up by the algae, corrections were made for the N and P removed

173

during sampling.

174

P and N removal followed zero order kinetics from the beginning of microalgae growth to P depletion

175

(from day 2 to 8). N removal remained mostly as zero order up to the end of the run, while P only

176

followed zero order kinetics after recuperation of the cells (from day 11 to 14).

177

RESULTS AND DISCUSSION

178

Microalgae screening

179

Three out of the six tested species successfully grew on AnBW, namely the green microalgae C.

180

vulgaris, S. obliquus and C. sorokiniana. The cyanobacteria species S. elongates, A. flos-aquae and S.

181

sp. showed no growth (Figure 1). The lack of growth by these cyanobacteria might be due to

182

photoinhibition just after inoculation30, 31 or due to something in the AnBW composition that inhibited

183

the cyanobacterial growth. This needs further investigation.

184

The three microalgae species that did successfully grow on AnBW reached biomass concentrations of

185

about 4 g dry weight. l-1 after 13 days (Table 2). Average biomass productivities over the entire

186

cultivation period were 0.28, 0.33 and 0.34 g dry weight. l-1.d-1 for C. vulgaris, C. sorokiniana and S.

187

obliquus, respectively. The biomass productivity was quite constant throughout the entire experiment

188

for each species, as can be concluded from the relatively linear increase in chlorophyll a fluorescence

189

over time (Figure 1).

190


Page 9 of 19


18

Chlorophyll a fluorescence

16 14 12 10 8 6 4 2 0 0

1

2

3

4

5

191

6 7 8 Time (d)

9 10 11 12 13 14

192

Figure 1. Chlorophyll a fluorescence of all algae species tested in time. C. sorokiniana (▲), C.

193

vulgaris (■), S. obliquus (◊), S. elongates (○), A. flos-aquae (+) and S. sp (×). Error bars represent

194

standard deviation between triplicates.

195

All three species showed an NH4+-N removal of about 30%, while the PO4-P removal varied greatly,

196

between 22 and 93% (Table 2). The NH4+-N removed from the medium was all taken up by the algae

197

biomass, with 102, 103 and 104 % of the removed N ending up in the biomass, as explained in the

198

Calculations section, for C. vulgaris, C. sorokiniana and S. obliquus respectively. The slightly higher

199

than 100% values was most likely due to measurement error. For the removed PO4-P, 119, 118 and

200

136 % ended up in the biomass for C. vulgaris, C. sorokiniana and S. obliquus respectively, indicating

201

that there must have been release of P from the suspended solids of the AnBW, as has been reported in

202

the literature6. Based on the initial TP (105.2 mg.l-1) it could be calculated that this release of P and

203

subsequent uptake by the algae was partial and some P must have remained in the solids by the end of

204

the experiment.

205

The removal rates of N and P for the three species were 13, 17 and 16 mg N.l-1.d-1 and 1.4, 2.1 and 5.9

206

mg P.l-1.d-1 for C. vulgaris, C. sorokiniana and S. obliquus respectively. These values are in

207

accordance with literature on microalgae growth under low light in various cultivation systems with

208

different geometries and different operating conditions, varying from 0.5 to 44 mg N.l-1.d-1 and from

209

1.8 to 9.4 mg P.l-1.d-1

16, 22, 32

. 9 ACS Paragon Plus Environment


Page 10 of 19

210

Table 2. Dry biomass concentration (Cx) ± standard deviation, C, N, and P molar ratios and % in algal

211

dry biomass, and N and P removal efficiencies, as explained in the Calculations section, at the end of

212

the experiments (day 13) from the AnBW, of three microalgae species.

Biomass

C. vulgaris

Biomass composition

Cx

C

N

P

g.l-1

Mol/mol P

C

Removal from AnBW

N

P

NH4+-N

mass fraction (% of dry biomass)

PO4-P

%

3.7 ± 0.2 93 16

1

40

7.7

1.1

26

22

C. sorokiniana 4.3 ± 0.2 90 15

1

42

8.0

1.2

30

39

S. obliquus

1

40

7.5

2.5

27

93

4.4 ± 0.1 42

7

213 214

Both C. vulgaris and C. sorokiniana showed N:P ratios similar to the Redfield ratio of 16:1, which is

215

commonly used as reference for microalgal species33 (Table 2). Scenedesmus obliquus on the other

216

hand, had a lower N:P ratio due to its higher P content. This high P content can point towards an

217

unusually high P-requirement of this species, or towards a phenomenon called luxury consumption.

218

During luxury consumption a nutrient is taken up at a much higher rate than the actual requirement of

219

the cells, a mechanism that evolved to store nutrients that are occasionally scarce or to consume scarce

220

nutrients faster than competitors34. This high P-content together with the high biomass productivity

221

resulted in 93% removal of the PO4-P by S. obliquus (Table 2), indicating that this species may be

222

very suitable for recovery of P from wastewaters with high PO4-P content, such as olive mill

223

wastewaters35.


Page 11 of 19


224

The carbon content found for the three species, 40 - 42% (Table 2), was lower than 46 - 50% usually

225

found in the literature36-38. These lower values could be due to a different biochemical composition of

226

the algal biomass grown on AnBW, as it has been reported that differences in macromolecules, such as

227

lipids, may have a relevant effect on the C content of organism 39.

228

In conclusion, these screening experiments show that various green microalgae can successfully grow

229

on the very nutrient rich AnBW medium, which is favourable for reaching high final biomass

230

concentrations. However due to the very high ammonia concentrations (about 1gN.l-1) the N:P ratio of

231

the AnBW is also very high (~30:1). This imposes an extra challenge to find the appropriate species,

232

operational conditions and reactor mode of operation to efficiently reach full P but also full N

233

removal. In an effort to reach this, batch experiments in flat-panel PBRs were done, using C.

234

sorokiniana.

235

N and P removal in controlled photobioreactors

236

Biomass growth together with PO4-P and NH4+-N removal was followed in three PBR replicates as

237

shown in Figure 2 (A). From day 9, the second part of the experiment took place, where the replicates

238

were operated as single batches (Figure 2 (B), (C) and (D)). PBR1 was kept without changes, PBR 2

239

received extra PO4-P addition and PBR 3 was stopped.

240

Nutrient replete cultivation phase

241

A lag phase of approximately two days was observed before microalgae growth commenced. During

242

this period, the PO4-P concentration increased, which was probably caused by solubilisation of the

243

inorganic PO4-P from the suspended solids of the AnBW (TP of 105.2±3.9 g·L-1) as a reaction to the

244

decrease of pH from 7.5 (AnBW) to 7 (set pH), similar to what has been reported earlier in the

245

literature6. The increase of soluble inorganic PO4-P was advantageous for a more favourable N:P

246

molar ratio of the AnBW (changing it from 30:1 to 20:1).



Page 12 of 19

180

1200

160

A

1000

120

800

100 600 80 60

400

NH4+-N (mg.l-1)

PO4-P (mg.l-1) algal hold-up (10-1ml.l-1)

140

40 200 20 0

0 1

2

3

4 5 6 Time (days)

120 100 80 60 PBR 1 PBR 2

40

C algal hold-up (10-1ml.l-1)

140

PBR 1 PBR 2

160

D

1000

140 120 100 80 60

800 PBR 1 PBR 2

600 400

40

200

20

20 0

9 10 11 12 13 14

9

1200

PO4-P (mg.l-1)

180 160

8

180

200

B

7

NH4+-N (mg.l-1)

0

0

0 9 10 11 12 13 14

9 10 11 12 13 14

247

Figure 2. PO4-P (●) and NH4+-N (■) removal from AnBW by C. sorokiniana and its growth,

248

presented as algal hold-up (∆), in time. (A) results from the three PBR replicates in the first 9 days;

249

(B) (C) and (D) from PBR 1 and PBR 2 (extra PO4-P addition at day 9) from day 9 to day 14. Error

250

bars in Figure (A) represent standard deviation between triplicates.

251

Once algae growth commenced (day 2), PO4-P was finished in 4 days (day 6) at a rate of 26 mg.l-1.d-1

252

and a removal yield on light of 12 mg PO4-P.mol photon-1. During that same 4-day period, nitrogen

253

removal occurred at a rate of 127 mg NH4+-N.l-1.d-1 and a removal yield on light of 56 mg NH4+-N.mol

254

photon-1. The N:P removal ratio in these 4 nutrient replete days (11:1) was much lower than the N:P 12 ACS Paragon Plus Environment

Page 13 of 19


255

ratio found for the C. sorokiniana during the screening experiments, suggesting that most likely PO4-P

256

was being stored as P-reserves within the cells. Indeed, this suggestion is confirmed by a virtually

257

unchanged NH4+-N removal rate after PO4-P depletion, from day 6 to 8 (Figure 2 (A)). Once the P-

258

reserves were depleted and the N:P removal ratio reached 16:1 (at day 8), which is identical to the

259

Redfield ratio and the ratio found for C. sorokiniana in the screening experiments, the N-removal rate

260

decreased substantially from 122 mg.l-1.d-1 (between day 2 and 8) to 41 mg.l-1.d-1 (between day 9 and

261

14) in PBR1 (Figure 2).

262

Over the 6-day period of constant N removal (day 2 to 8), an NH4+-N removal rate of 122 mg.l-1.d-1

263

was observed, corresponding to an N removal yield on light of 30 mg NH4+-N.mol photon-1. The

264

average PO4-P removal rate over this same period was 17 mg.l-1.d-1, corresponding to a P removal

265

yield on light of 4.2 mg PO4-P.mol photon-1. Based on these N and P removal rates, and on the N and

266

P contents observed during the screening experiments (8 and 1.2 % respectively, Table 2), the biomass

267

concentration at day 8 was estimated to have been around 9 g dry weight.l-1. Similarly, the biomass

268

yield on light was estimated at 0.4 g dry weight.mol photon-1, which is just below the values reported

269

for growth on human urine (0.55 and 0.7 g dry weight.mol photon-1)18 and about half of the optimal

270

values reported for C. sorokiniana 40, 41. Because the temperature and pH were kept close to optimal

271

for this species, the low biomass yield in the present experiments is most likely explained by a

272

suboptimal light regime and/or the characteristics of the AnBW. We can however conclude that under

273

nutrient replete conditions C. sorokiniana was quite efficient in removing NH4+-N and PO4-P from

274

AnBW. However, this efficiency changed when the P-reserves were depleted.

275

Nutrient-limited cultivation phase – effect of P addition

276

To study the effect of P-limitation on the nitrogen removal efficiency, 75mg PO4-P.l-1 was added to

277

PBR 2 at day 9 of the experiment. PBR 1 was continued without any changes and PBR 3 was stopped

278

due to operational problems. This addition did not significantly improve the removal rate of NH4+-N

279

from PBR2 compared to PBR1, with both reactors reaching full N-removal at day 14 of the



Page 14 of 19

280

experiment. The nitrogen removal rates between day 9 and 14 were 41 mg NH4+-N.l-1.d-1 for PBR1

281

and 40 mg NH4+-N.l-1.d-1 for PBR2 (Figure 2).

282

The lack of effect on N uptake could have been due to a too late resupply of PO4-P. Namely, at the

283

moment of replenishment the microalgae had already been deprived of P for 1 day, which might have

284

forced the cells to enter the stationary phase. Such a physiological state could have resulted in low

285

recovery rates upon P-resupply, resulting in low growth rates and consequently low nutrient removal

286

rates. Indeed, PO4-P consumption only started two days after P-resupply. Once PO4-P consumption

287

commenced at day 11, the P removal rate was constant until P was depleted at day 14. The P removal

288

rate during this period was similar to that observed between day 2 and 6 (28 vs. 26 mg PO4-P.l-1.d-1,

289

respectively).

290

The addition of PO4-P did however affect cell growth in terms of algal hold-up (Figure 2B). Without

291

this P-addition, the continued NH4+-N removal during P-limitation (from day 8 to 14) corresponded to

292

a rather varying algal hold-up, averaging around 12 ml cell volume per liter reactor volume which is

293

similar to the hold-up at the onset of P-limitation at day 8 (Figure 2). P-resupply, however, caused the

294

algal hold-up to reach 19 ml.l-1 at the end of the experiment (Figure 2B). These differences suggest a

295

totally different physiology, despite the similar N removal rates.

296

Overall experiment

297

The severe drop in removal rates at the onset of P-limitation (at day 8) finally yielded overall nutrient

298

removal rates of 82 and 76 mg NH4+-N.l-1.d-1 and 9 and 15 mg PO4-P.l-1.d-1 for PBR 1 and 2

299

respectively (Table 3). The higher PO4-P removal rate for PBR 2 was due to the addition of PO4-P at

300

day 9. These overall removal rates corresponded to removal yields on light of 13.5 mg NH4+-N.mol

301

photon-1 and 1.5 mg PO4-P.mol photon-1. The biomass concentration at the end of the experiment

302

reached between 10.5 and 12.1 g dry weight.l-1 (Table 3), which corresponds to a quite low biomass

303

yield on light of 0.16 g dry weight.mol photon-1.

304

This high final biomass density makes this process attractive for large-scale application, because it

305

will reduce the costs of algae harvesting. On the other hand the low overall yields on light for biomass 14 ACS Paragon Plus Environment

Page 15 of 19


306

production and nutrient removal reached will increase photobioreactor investment costs because

307

removal yields on light directly relate to the surface of photobioreactor-area required to treat a certain

308

amount of wastewater. A consideration could be to stop the process earlier while the yields on light are

309

still high and use alternative methods for removal of the remaining N, such as partial nitritation and

310

anammox42.

311

In the process described above, for instance, stopping at day 8 would have resulted in a 2.2-fold higher

312

NH4+-N removal yield on light compared to stopping at day 14, with 100% of the PO4-P and 76% of

313

the NH4+-N being removed after these 8 days compared to full removal of both nutrients at day 14.

314

The estimated biomass concentration at day 8 was about 9 g dry weight.l-1, which is still favourable for

315

efficient harvesting. Whether the incomplete N-removal is acceptable, or whether the remaining NH4+-

316

N can be removed more efficiently than in the process described above, is dependent on the treatment

317

targets and the extent of future improvement of the above described process.

318

A possible solution for reaching full N and P removal at a faster rate and at a higher yield on light,

319

could be to use or add other microalgae with even higher N:P uptake ratios. It is known that depending

320

on the growth conditions, algae may obtain N:P ratios much higher as compared to the Redfield ratio

321

(16:1)12, 43-45. More specifically, cellular N:P ratios of algae increase with reduced growth, as well as

322

under resource limitation (particularly P)39, 44, 46. This will be investigated in future experiments.

323 324

Table 3. Biomass composition, removal % and removal rates of NH4+-N and PO4-P from AnBW,

325

biomass concentration and production rate and yield of biomass on light energy of C. sorokiniana at

326

the end of the linear growth and end of the experiment. Day 2 – 8 Description

Unit

Day 2 – 14

PBR 1 - 3

PBR 1

PBR 2

125:19:1

73:12:1a

mol.molP-1

C:N:P

n.d.

%

C,N, P

n.d.

Biomass composition 43, 7.7, 0.9 41,7.4, 1.4a



NH4+-N Removal from AnBW

76.1 ± 6.6

98.4

98.9

100

100

100

NH4+-N 121.9 ± 13.3

81.8

76.4

PO4-P

16.8 ± 0.9

8.9

14.9a

9.1 ± 1.0b

10.5

12.1

NH4+-N

30.3 ± 3.4

13.7

13.2

PO4-P

4.2 ± 0.2

1.5

2.6 a

0.4 ± 0.04

0.15

0.17

% PO4-P

Removal rates

Biomass concentration

Removal yield on light

Page 16 of 19

-1

-1

mg.l .d

g DW.l-1 c

mg.mol photon

Yield of biomass on light g.mol photon-1

-1

327

a

Values different for PBR 2 in relation to PBR1 due to addition of 75mg PO4-P.l-1 at day 9.

328

b

Estimated from N removal from day 2 to 8 and final N content in biomass observed during the

329

screening experiments (8 %, Table 2)

330

c

DW = dry weight.

331 332

In conclusion, C. sorokiniana can remove all PO4-P within 4 days and all NH4+-N within 12 days, at

333

the light intensities and photobioreactors used in this experiment. Favourable removal yields were

334

achieved as long as N and P did not become limiting. These cultivations resulted in high microalgal

335

biomass concentrations of 10.5 and 12.1 g DW.l-1. We believe our findings form a solid basis for

336

further exploration of this technology for full-scale applicability.

337 338

ACKNOWLEDGMENTS

339

We thank Nico Helmsing, Suzanne Wiezer and Hans Meijer for their technical support. We also like

340

to thank Bas Ibelings and Dedmer van de Waal for useful discussions. Funding came from the

341

Netherlands Enterprise Agency (RVO), Ministry of Economic Affairs, The Netherlands (Innowator

342

project).


Page 17 of 19


343

REFERENCES

344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390

1. Zeeman, G.; Kujawa, K.; de mes, T.; Hernandez Leal, L.; De Graaff, M. S.; Abu-Ghunmi, L. N. A. H.; Mels, A. R.; Meulman, B.; Temmink, H.; Buisman, C. J. N.; Van Lier, J. B.; Lettinga, G., Anaerobic treatment as a core technology for energy, nutrients and water recovery from source-separated domestic waste (water). Water Science and Technology 2008, 57, (8), 1207 - 1212. 2. Zeeman, G.; Kujawa-Roeleveld, K., Resource recovery from source separated domestic waste(water) streams; full scale results. Water Science and Technology 2011, 64, (10), 1987-1992. 3. Cordell, D.; Rosemarin, A.; Schröder, J. J.; Smit, A. L., Towards global phosphorus security: A systems framework for phosphorus recovery and reuse options. Chemosphere 2011, 84, (6), 747-758. 4. Kujawa-Roeleveld, K.; Zeeman, G., Anaerobic Treatment in Decentralised and SourceSeparation-Based Sanitation Concepts. Reviews in Environmental Science and Biotechnology 2006, 5, (1), 115-139. 5. Mihelcic, J. R.; Fry, L. M.; Shaw, R., Global potential of phosphorus recovery from human urine and feces. Chemosphere 2011, 84, (6), 832-839. 6. de Graaff, M. S.; Temmink, H.; Zeeman, G.; Buisman, C. J. N., Energy and phosphorus recovery from black water. Water Science and Technology 2011, 63, (11), 2759-2765. 7. Wijffels, R. H.; Barbosa, M. J., An Outlook on Microalgal Biofuels. Science 2010, 329, (5993), 796-799. 8. Mulbry, W.; Westhead, E. K.; Pizarro, C.; Sikora, L., Recycling of manure nutrients: use of algal biomass from dairy manure treatment as a slow release fertilizer. Bioresource Technology 2005, 96, (4), 451-458. 9. Godos, I. d.; Vargas, V. A.; Blanco, S.; González, M. C. G.; Soto, R.; García-Encina, P. A.; Becares, E.; Muñoz, R., A comparative evaluation of microalgae for the degradation of piggery wastewater under photosynthetic oxygenation. Bioresource Technology 2010, 101, (14), 5150-5158. 10. Levine, R. B.; Costanza-Robinson, M. S.; Spatafora, G. A., Neochloris oleoabundans grown on anaerobically digested dairy manure for concomitant nutrient removal and biodiesel feedstock production. Biomass and Bioenergy 2011, 35, (1), 40-49. 11. Mallick, N., Biotechnological potential of immobilized algae for wastewater N, P and metal removal: A review. BioMetals 2002, 15, (4), 377-390. 12. Wang, L.; Min, M.; Li, Y.; Chen, P.; Chen, Y.; Liu, Y.; Wang, Y.; Ruan, R., Cultivation of Green Algae Chlorella sp. in Different Wastewaters from Municipal Wastewater Treatment Plant. Applied Biochemistry and Biotechnology 2010, 162, (4), 1174-1186. 13. Wilkie, A. C.; Mulbry, W. W., Recovery of dairy manure nutrients by benthic freshwater algae. Bioresource Technology 2002, 84, (1), 81-91. 14. Abeliovich, A., Handbook of microalgal culture, biotechnology and applied phycology. Blackwell Publishing: Oxford, 2005. 15. Christenson, L.; Sims, R., Production and harvesting of microalgae for wastewater treatment, biofuels, and bioproducts. Biotechnology Advances 2011, 29, (6), 686-702. 16. Pires, J.; Alvim-Ferraz, M.; Martins, F.; Simoes, M., Wastewater treatment to enhance the economic viability of microalgae culture. Environmental Science and Pollution Research 2013, 20, (8), 5096-5105. 17. Abdel-Raouf, N.; Al-Homaidan, A. A.; Ibraheem, I. B. M., Microalgae and wastewater treatment. Saudi Journal of Biological Sciences 2012, 19, (3), 257-275. 18. Tuantet, K.; Temmink, H.; Zeeman, G.; Janssen, M.; Wijffels, R. H.; Buisman, C. J. N., Nutrient removal and microalgal biomass production on urine in a short light-path photobioreactor. Water Research 2014, 55, (0), 162-174. 19. Redfield, A., The biological control of chemical factors in the environment. American Scientist 1958, 46, 205–221.



391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442

Page 18 of 19

20. Bährs, H.; Steinberg, C., Impact of two different humic substances on selected coccal green algae and cyanobacteria—changes in growth and photosynthetic performance. Environmental Science and Pollution Research 2012, 19, (2), 335-346. 21. Pouneva, I. D., Effect of Humic Substances on the Growth of Microalgal Cultures. Russian Journal of Plant Physiology 2005, 52, (3), 410-413. 22. Marcilhac, C.; Sialve, B.; Pourcher, A.-M.; Ziebal, C.; Bernet, N.; Béline, F., Digestate color and light intensity affect nutrient removal and competition phenomena in a microalgal-bacterial ecosystem. Water Research 2014, 64, (0), 278-287. 23. Kilham, S.; Kreeger, D.; Lynn, S.; Goulden, C.; Herrera, L., COMBO: a defined freshwater culture medium for algae and zooplankton. Hydrobiologia 1998, 377, (1-3), 147-159. 24. Breuer, G.; Lamers, P. P.; Martens, D. E.; Draaisma, R. B.; Wijffels, R. H., Effect of light intensity, pH, and temperature on triacylglycerol (TAG) accumulation induced by nitrogen starvation in Scenedesmus obliquus. Bioresource Technology 2013, 143, (0), 1-9. 25. Cuaresma Franco, M.; Buffing, M.; Janssen, M.; Vílchez Lobato, C.; Wijffels, R., Performance of Chlorella sorokiniana under simulated extreme winter conditions. Journal of Applied Phycology 2012, 24, (4), 693-699. 26. APHA, Standard Methods for the Examination of Water and Wastewater. 21st edn ed.; American Public Health Association: Washington DC, 2005. 27. Fernandes, T. V.; Keesman, K. J.; Zeeman, G.; van Lier, J. B., Effect of ammonia on the anaerobic hydrolysis of cellulose and tributyrin. Biomass and Bioenergy 2012, (47), 316 - 323. 28. Santos, A. M.; Wijffels, R. H.; Lamers, P. P., pH-upshock yields more lipids in nitrogen-starved Neochloris oleoabundans. Bioresource Technology 2014, 152, (0), 299-306. 29. Cuaresma, M.; Janssen, M.; Vílchez, C.; Wijffels, R. H., Horizontal or vertical photobioreactors? How to improve microalgae photosynthetic efficiency. Bioresource Technology 2011, 102, (8), 5129-5137. 30. Campbell, D.; Hurry, V.; Clarke, A. K.; Gustafsson, P.; Öquist, G., Chlorophyll Fluorescence Analysis of Cyanobacterial Photosynthesis and Acclimation. Microbiology and Molecular Biology Reviews 1998, 62, (3), 667-683. 31. Ibelings, B. W.; Kroon, B. M. A.; Mur, L. R., Acclimation of Photosystem II in a Cyanobacterium and a Eukaryotic Green Alga to High and Fluctuating Photosynthetic Photon Flux Densities, Simulating Light Regimes Induced by Mixing in Lakes. New Phytologist 1994, 128, (3), 407-424. 32. Kim, S.; Park, J. E.; Cho, Y. B.; Hwang, S. J., Growth rate, organic carbon and nutrient removal rates of Chlorella sorokiniana in autotrophic, heterotrophic and mixotrophic conditions. Bioresource Technology 2013, 144, 8-13. 33. Redfield, A., On the proportion of organic derivates in sea water and their relation to the composition of plankton. In James Johnstone Memorial Volume, Daniel, R. J., Ed. University Press of Liverpool: 1934; pp 177-192. 34. Powell, N.; Shilton, A. N.; Pratt, S.; Chisti, Y., Factors Influencing Luxury Uptake of Phosphorus by Microalgae in Waste Stabilization Ponds. Environmental Science & Technology 2008, 42, (16), 5958-5962. 35. Kapellakis, I.; Tzanakakis, V. A.; Angelakis, A. N., Land Application-Based Olive Mill Wastewater Management. Water 2015, 7, (2), 362-376. 36. Kumar, K.; Dasgupta, C. N.; Das, D., Cell growth kinetics of Chlorella sorokiniana and nutritional values of its biomass. Bioresource Technology 2014, 167, 358-366. 37. Bhattacharjee, A.; Ghosh, S. K.; Ghosh, D.; Ghosh, S.; Maiti, M. K.; Sen, S. K., Identification of a heat-stable palmitoyl/oleoyl specific acyl-acyl carrier protein thioesterase in developing seeds of Madhuca butyracea. Plant Science 2002, 163, (4), 791. 38. Tang, D.; Han, W.; Li, P.; Miao, X.; Zhong, J., CO2 biofixation and fatty acid composition of Scenedesmus obliquus and Chlorella pyrenoidosa in response to different CO2 levels. Bioresource Technology 2011, 102, (3), 3071-3076. 39. Sterner, R. W.; Elser, J. J., Ecological Stoichiometry: The Biology of Elements from Molecules to the Biosphere. Princeton University Press: Princeton, NJ, 2002. 18 ACS Paragon Plus Environment

Page 19 of 19

443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461


40. Kliphuis, A. M. J.; de Winter, L.; Vejrazka, C.; Martens, D. E.; Janssen, M.; Wijffels, R. H., Photosynthetic efficiency of Chlorella sorokiniana in a turbulently mixed short light-path photobioreactor. Biotechnology Progress 2010, 26, (3), 687-696. 41. Zijffers, J.-W.; Schippers, K.; Zheng, K.; Janssen, M.; Tramper, J.; Wijffels, R., Maximum Photosynthetic Yield of Green Microalgae in Photobioreactors. Marine Biotechnology 2010, 12, (6), 708-718. 42. de Graaff, M. S.; Temmink, H.; Zeeman, G.; van Loosdrecht, M. C. M.; Buisman, C. J. N., Autotrophic nitrogen removal from black water: Calcium addition as a requirement for settleability. Water Research 2011, 45, (1), 63-74. 43. Francoeur, S. N.; Biggs, B. J. F.; Smith, R. A.; Lowe, R. L., Nutrient Limitation of Algal Biomass Accrual in Streams: Seasonal Patterns and a Comparison of Methods. Journal of the North American Benthological Society 1999, 18, (2), 242-260. 44. Klausmeier, C. A.; Litchman, E.; Daufresne, T.; Levin, S. A., Optimal nitrogen-to-phosphorus stoichiometry of phytoplankton. Nature 2004, 429, (6988), 171-174. 45. Loladze, I.; Elser, J. J., The origins of the Redfield nitrogen-to-phosphorus ratio are in a homoeostatic protein-to-rRNA ratio. Ecology Letters 2011, 14, (3), 244-250. 46. Hillebrand, H.; Steinert, G.; Boersma, M.; Malzahn, A.; Meunier, C. L.; Plum, C.; Ptacnik, R., Goldman revisited: Faster-growing phytoplankton has lower N : P and lower stoichiometric flexibility. Limnology and Oceanography 2013, 58, (6), 2076-2088.

462


Closing Domestic Nutrient Cycles Using Microalgae - ACS Publications

Recommend Documents